The Effect And Molecular Mechanisms Of MiR-22on Adipogenic And Osteogenic Differentiation Of Human Adipose Tissue-derived Mesenchymal Stem Cells

Mesenchmal stem cells (MSCs) are a plutipotent cell type that can be differentiated into either adipocytes or osteoblasts. A mutually exclusive relationship exists between adipogenesis and osteogenesis due to the derivation from the common progenitor cell. Multiple transcription factors and signaling pathways have been reported to regulate adipogenic or osteogenic differentiation respectively, yet the molecular mechanism underlying the cell fate alteration between adipogenesis and osteogenesis still remains to be illustrated. The balance between adipocyte formation and osteoblast formation is associated with metabolic regulation and homeostasis maintaining in human tissue. Once the balance between adipogenesis and osteogenesis is disrupted, various bone-related diseases like osteoporosis will occur. Hence, investigation of the balance between adipogenic and osteogenic differentiation is of significant importance for illustration of the molecular mechanism underlying differentiation of MSCs, as well as for elucidation of pathogenesis and development of therapies for bone-related diseases.MicroRNAs (miRNAs), the endogenously expressed10-25bp non-protein-coding RNAs, are important regulators in diverse biological processes by repressing protein expression of their targets. In this study, we are going to investigate the effect and mechanisms of miR-22on adipogenic and osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hADMSCs).In the first part of this study, we tried to discuss the role of miR-22in adipogenic and osteogenic differentiation of hADMSCs. Firstly, we examined the endogenous expression of miR-22in hADMSCs during adipogenic and osteogenic differentiation and found that miR-22decreased during adipogenesis but increased during osteogenesis, indicating the dual impact of miR-22in regulating adipo/osteogenic differentiation of hADMSCs. By transfection of miR-22mimics into hADMSCs, we found that overexpression of miR-22could inhibit lipid droplets accumulation and repress the expression of the key adipogenic transcription factors (PPARy, C/EBPa) and adipogenic-specific genes (LPL, FABP4). Besides, enhanced alkaline phosphatase activity and matrix mineralization, as well as increased expression of osteo-specific genes was observed due to overexpression of miR-22. Taken together, the results above indicated that miR-22could inhibit adipogenic differentiation and promote osteogenic differentiation of hADMSCs.In the second part, we tried to identify the downstream target genes of miR-22. We searched for potential downstream targets of miR-22by two different miRNA target predition databases TargetScan and miRanda, and picked out7target genes that are directly or indirectly involved in adipo/osteognic differentiation as "candidate targets" We then performed dual luciferase report assay and identified that miR-22could repress post transcription via interaction with HDAC6mRNA3'UTR. Results of Western Blot and Real Time PCR suggested that miR-22could down-regulate HDAC6protein expression without affecting the expression levels of its mRNA. Taken together, HDAC6was identified as a downstream target of miR-22. We then focsed on discussing the impact of HDAC6on adipogenic and osteogenic differentiation of hADMSCs in the next part of our study in order to investigate the molegular mechanisms of miR-22-mediated regulation of adipogenesis and osteogenesis of hADMSCs.In the third part, we tried to discuss the downstream molecular mechanisms of miR-22-mediated regulation of adipogenic and osteogenic differentiation through the investigation of the effect of HDAC6on adipocyte and osteoblast formation of hADMSCs. Firstly, we found HDAC6was increased during adipogenesis while decreased during osteogenesis via both Western Blot and Real Time PCR examination. Inhibition of endogenous HDAC6by small interfering RNAs (siRNAs) suppressed adipogenesis and stimulated osteogenesis, consistent with the effect of miR-22overexpression in hADMSCs. Taken together, the results above indicated HDAC6as a promoter of adipogenic differentiation but a repressor of osteogenic differentiation in hADMSCs.In conclusion, our results suggested that miR-22acted as a critical regulator of balance between adipogenic and osteogenic differentiation of hADMSCs by repressing its target HDAC6.